'Molecular Portals' in Brain Cells Identified

DURHAM, N.C. -- Infinitesimal particles of gold have enabled neurobiologists to track down key molecules in the machinery of "entry points" in neurons -- offering clues to the organization of a region that has thus far remained largely unknown neuronal territory.

The researchers -- from Duke University Medical Center and the University of North Carolina -- used electron microscopy to locate molecules tagged with targeted antibodies attached to gold particles -- rendering the molecules' precise location visible.

The findings by the researchers, led by Michael Ehlers, M.D., of Duke and Richard Weinberg, Ph.D., of the University of North Carolina at Chapel Hill, were published online Aug. 22, 2004, in the journal Nature Neuroscience. Other co-authors are Bence Rácz of UNC and Thomas Blanpied, Ph.D., of Duke. The research was sponsored by the National Institutes of Health, the National Alliance for Research on Schizophrenia and Depression, Christopher Reeve Paralysis Foundation and the Broad Foundation.

Their studies aimed to understand how receptors on the surface membranes of nerve cells undergo a recycling process called endocytosis, in which the receptors are drawn into the interior of the neurons to be recycled.

These receptors are proteins that are activated by bursts of signaling chemicals, called neurotransmitters, launched from another, transmitting neuron. Such activation triggers a nerve impulse in the receiving neuron. Changes in the strength of a neuron's response to such chemical signals depend on the number of receptors on the dendritic spine surface. And the strength of such connections is key to establishing the neural pathways through the brain that are the basis of learning and memory.

The neurotransmitter "receiving stations" on the neuron are mushroom-shaped dendritic spines that festoon its surface. The signaling regions between neurons are known as synapses, and the receiving membrane on the dendritic spine is known as the postsynaptic membrane.

A key mystery about dendritic spines, said Ehlers, has been where on their surface such recycling of receptors takes place

"It has been known for some time that signal reception takes place in a small region of the spine membrane known as the postsynaptic density," he said. "But the postsynaptic density comprises only 15 percent of the membrane area. What happens in the remaining 85 percent of the spine's membrane has been almost completely unknown," he said.

"One way that connections in our brains are weakened is by removing receptors from synapses, but where this removal occurs has been unclear. Defining this 'microanatomy' of dendritic spines is thus quite fundamental to understanding how neural connections are formed and restructured as our brains develop, change, and age," he said.

According to Ehlers, it has been believed that receptors to be recycled "uncouple" from the postsynaptic density and move across the fatty membrane to an "endocytic zone." In this unidentified zone, molecular machinery attaches to the receptor, draws it into a bubble-like vesicle and transports it to machinery where it is either recycled or destroyed.

To attempt to map such zones, the researchers decided to trace the precise location of three key molecules known to play central roles in endocytosis:

-- clathrin, the protein that stitches together like a soccer ball to create the vesicle that buds from the membrane,

-- AP-2, the adaptor molecule that grabs onto receptor cargo and attaches it to clathrin, and

-- dynamin, the protein that drives the machinery that pinches the vesicle off from the cell membrane, freeing it to travel to the recycling machinery.

The researchers attached gold particles to antibodies that specifically targeted each of these proteins, and used electron microscopy to search for these molecules in rat brain tissue. Their tracking revealed that each of the molecules concentrates in specific lateral zones of the spines.

"If you think of the spine as a roughly spherical structure with the synapse at 12 o'clock, we found that these endocytic molecules concentrate at zones at 3 o'clock and 6 o'clock," said Ehlers. He said that these concentrations mark the spots at which the membrane is internalized by endocytosis and the receptors drawn in. And even when the spines are larger or smaller, the distances expand or shrink so the zones stay at the same relative positions.

"While we still don't fully understand how this zone is established or how molecules move through this zone into the cell interior, with these findings, we are beginning to see a level of organization that we didn't know existed," said Ehlers.

"These findings imply a hidden level of organization on the dendrite that's yet to be revealed," said Ehlers. "This specialized endocytic zone is only the second known membrane specialization in dendritic spines."

What's more, he said, the existence of zones in the postsynaptic membrane mirrors a similar organization known to exist on the "presynaptic" terminals on the transmitting neurons that launch bursts of neurotransmitter.

Ehlers also said that the findings of organization on dendritic spines could have broader implications in understanding signaling between nerve cells.

"It's well known that many kinds of receptors -- not just neurotransmitter receptors -- undergo downregulation by endocytosis," said Ehlers. "These include receptors involved in learning and memory, tolerance to medications, or reactions to drugs of abuse. So, I think our findings regarding the spatial organization of endocytosis will be relevant in understanding a wide range of such processes."